Principal Investigators: GREALLY, J.M., LEVY, M. Project abstract IN VIVO IMAGING OF X INACTIVATION. We propose to develop a system for in vivo imaging of the epigenetic regulatory processes involved in X chromosome inactivation. X inactivation is a well-studied paradigm of epigenetic gene regulation, involving the silencing of the majority of the genes on one X chromosome in female cells, part of the process of dosage compensation in mammals. A number of epigenetic regulatory processes have been found to contribute to the inactivation process, which when imaged using immunofluorescence on fixed cells generate a signal throughout the chromosome territory of the inactive X. The robustness of this signal makes X inactivation an attractive system for the development of in vivo imaging approaches. The inactive X is characterized by the presence of repressive post-translational histone modifications such as histone H3 lysine 9 trimethylation (H3K9me3) and H3K27me3, modifications established by polycomb group proteins which, when mutated, are associated with the failure of X inactivation. There are, however, other regulatory mediators implicated with functions that are less obviously related to the establishment of these chromatin states, functions such as helicase activity, RNA-binding, matrix-attachment region DNA-binding, or those functions associated with chromosomal structural maintenance motifs. As a means of understanding how each component of the X inactivation system interacts functionally, an in vivo system would allow the observation of sequential localization of the protein mediators and histone modifications to the inactivating X chromosome, thus establishing a likely hierarchy of regulation in this complex epigenetic process. In order to develop such a system, a number of areas of expertise need to be assembled. The project starts with the in vitro generation of histone peptides (and eventually entire reconstituted nucleosomes) with methylation and ubiquitination marks (David Allis and Tom Muir, Rockefeller University) that are then used for in vitro selection by co-PI Matthew Levy (Einstein) to create RNA aptamers specifically binding to these post- translational modifications. These aptamers are then linked in an expression construct to RNA hairpins bound by fluorescently-tagged phage coat proteins, a system pioneered by co-investigator Robert Singer (Einstein) as a means of tracking RNA in vivo in transcription studies. This project represents the first use of the same system for epigenetic studies. The cell type in which the system will be optimized will be a female mouse embryonic stem cell line, allowing not only X inactivation studies but also the broader use of this system in pluripotent cells when made available to the scientific community. The X inactivation studies will be facilitated by the development of fluorescent tags for the candidate protein mediators of X inactivation (Edith Heard, Institut Curie, Paris, France). The project is thus based on a strong and multifaceted foundation of expertise and resources. PUBLIC HEALTH RELEVANCE: Principal Investigators: GREALLY, J.M., LEVY, Matthew Project narrative IN VIVO IMAGING OF X INACTIVATION. In this project, we propose to develop a system that will allow us to see within the cell nucleus how processes that are important for switching genes on and off are physically interacting. We are using a dramatic example of gene regulation, the inactivation of one of the X chromosomes in female cells. We already know that there are numerous proteins that are involved with inactivation of the X chromosome, and we recognize that the inactive X is marked by the addition of certain chemical groups to the proteins contained within that chromosome. What is not apparent is how each of these regulatory mediators controls or is controlled by the others involved. At present, we need to kill cells to see where these regulatory mediators are located within the cell, but if we were able to watch them in living cells we could determine the order in which these regulators exert their effects, thus getting an indication of the regulatory mechanism in this process. We propose to combine a number of technologies that have not been brought together previously in order to be able to watch how these regulators interact in the living cell. We will use mouse embryonic stem cells, a cell type that can turn into most cell types in the body, so the resources we develop will be more generally useful to the scientific community and will not be restricted to the study of X inactivation. We will also use a new technology involving nucleic acid structures called aptamers, selecting them for their ability to bind specifically to the chemical groups or the proteins of interest. The investigators involved represent leaders in the fields of X chromosome inactivation, chromatin biology, live cell imaging and aptamer technology, and the goal is not only to gain insights into X chromosome inactivation but also to develop resources that can be used by the broader scientific community.